Degradation of cellular proteins is required for normal maintenance of cellular function, including proliferation, differentiation, and cell death. The irreversible nature of proteolysis makes it well-suited to serve as a regulatory switch for controlling unidirectional processes. This principle is evident in the control of the cell cycle, where initiation of DNA replication, chromosome segregation, and exit from mitosis are triggered by the destruction of key regulatory proteins.
One of the major pathways to regulate proteins posttranslationally is ubiquitin-dependent proteolysis. The first step in selective degradation is the ligation of one or more ubiquitin molecules to a protein substrate. Ubiquitination occurs through the activity of ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin-protein ligases (E3), which act sequentially to catalyze the attachment of ubiquitin to lysine residues of substrate proteins (See Ciechanover A., et al., BioEssays, 22:442–451 (2000)). The E3s confer specificity to ubiquitination reactions by binding directly to substrate.
There is a need in the art to provide methods and compositions useful for regulating protein activities, especially in vivo. There is also a need in the art to provide methods for identifying proteins involved in a predetermined function or phenotype.